Mechanisms of Visual Signaling

描述（由申请人提供）：这项工作的长期目标是阐明脊椎动物光响应信号转导的分子机制。在本申请中，我们提出了实验来表征恢复素与视紫红质激酶的相互作用。这个建议是基于初步实验，从最后一批期间，其中一个以前未知的构象recoverin形成时，钙激活recoverin结合到视紫红质激酶或激酶的N-末端肽片段的完整RGS结构域。重要的是，这种新的构象似乎是目前作为一个次要的物种（RvCaB）的平衡混合物（RvCaA/RvCaB）后，形成的Ca 2+结合到recoverin，即使在没有激酶。我们的方法将是使用生物物理技术（主要是NMR光谱）和生化分析与纯化的成分在体外条件下的组合，以解开这两种蛋白质的相互作用的结构和作用机制的细节。该提案有三个具体目标：1。完成RvCaB的结构表征; 2.通过诱变研究来探索哪些氨基酸残基的恢复有助于RvCaB的稳定;和3.确定恢复素/视紫红质激酶复合物的原子细节结构，最初关注激酶的RGS结构域。公共卫生相关性：这项研究将在三个方面产生影响：1。了解参与蛋白质-蛋白质相互作用的信号分子的基本机制;理解癌症相关视网膜病变和细胞增殖的异常恢复蛋白表达;和3.了解神经元钙传感器蛋白的一般机制。

DESCRIPTION (provided by applicant): The long-term goal of this work is to elucidate molecular mechanisms of vertebrate photoresponse signal transduction. In the current application we propose experiments to characterize the interaction of recoverin with rhodopsin kinase. This proposal is based upon preliminary experiments from the last grant period in which a previously unknown conformation of recoverin was formed when Ca2+-activated recoverin was bound to either the full RGS domain of rhodopsin kinase or an N-terminal peptide fragment of the kinase. Importantly, this new conformation appears to be present as a minor species (RvCaB) in an equilibrium mixture (RvCaA/RvCaB) formed upon the binding of Ca2+ to recoverin even in the absence of the kinase. Our approach will be to use a combination of biophysical techniques (primarily NMR spectroscopy) and biochemical analyses with purified components under in vitro conditions to unravel details of both the structure and mechanism of action for the interaction of these two proteins. There are three Specific Aims in this proposal: 1. To complete structural characterization of RvCaB; 2. To probe through mutagenesis studies which amino acid residues in recovern contribue to stabilization of RvCaB; and 3. To determine in atomic detail the structure of the recoverin/rhodopsin kinase complex with initial focus on the RGS domain of the kinase. PUBLIC HEALTH RELEVANCE: This study will have an impact in three areas: 1. Understanding fundamental mechanisms of signaling molecules involved in protein-protein interactions; 2. Understanding of aberrant recoverin expression for cancer-associated retinopathy and cell proliferation; and 3. Understanding general mechanisms of neuronal calcium sensor proteins.